The mechanisms of allosteric influence on the activity of fumarase and phosphofructokinase will be studied by measuring the free energy of interaction between allosteric ligand and substrate (Fax), the influence onmaximal velocity (W), and the dissociation constant of the allosteric ligand from free enzyme (Rix). An approach has been developed to determine these parameters from kinetic experiments in a non-cooperative enzyme such as pig heart fumarase, and it has been used to establish the allosteric nature of the tripolyphosphate inhibition of this enzyme. The molecular features of both the enzyme andligand responmsible for this allosteric interaction will be studied by syhstematically varying the structure and charge of the ligand and by varying the pH and dielectric contant of the solvent. These studies should provide considerable insight into the actual molecular interactions which occur within the tertiary structure oftheprotein-ligand complex to produce the "conformational change" associated with the allosteric modification of catalytic efficiency. Fax, W, and Kix will be measured for a variety of allosteric ligands of rat liver phosphofructokinase through a combination of kinetic and binding experiments. The ligand-ligand interactions manifest in a system of three ligands (fructose 6-phosphate, ATP, and activator) will also be systematically evaluated as will themechanistic origins of the unusally high degree of cooperativity associated with the fructose 6-phosphate saturation profile. These results will be compared to those obtained from experiments performed with rabbit muscle phosphofructokinase in an effort to more accurately describe the functional difference between these two isozymes. In addition, these studies should greatly improve our understanding of the causes of the complex regulatoryh behavior which is exhibited by this enzyme and which in turn controls in large part the pace and direction of carbohydrate metabolism in both liver and muscle.